Torben Johansen

The utilization of adenosine triphosphate in rat mast cells during histamine release induced by anaphylactic reaction and compound 48/80

SummaryThe ATP content of rat peritoneal mast cells has been studied in relation to histamine release induced by compound 48/80 and antigen-antibody (anaphylactic) reaction in vitro. When the ATP content of actively sensitized mast cells was reduced to different levels by oligomycin, a good correlation was obtained between the ATP levels and the amounts of histamine released by the anaphylactic reaction. A similar linear relation has previously been demonstrated between the ATP levels of mast cells and histamine release induced by compound 48/80. The ATP content of mast cells was also studied at different intervals after the exposure of the cells to antigen or compound 48/80. No significant change in the ATP content was observed in untreated mast cells during the short period when histamine release occurs. If, however, the mast cells were preincubated with oligomycin or 2-deoxyglucose to reduce the rate of ATP synthesis while a large part of the histamine release remained unaffected—a decrease in the ATP content could be demonstrated in close time relation to both anaphylactic and compound 48/80-induced histamine release. The observations indicate an increased utilization of ATP in mast cells during the release process.

Role of the Na+/K+‐ATPase in regulating the membrane potential in rat peritoneal mast cells

1 The aim of this study was to investigate the effect of the Na+/K+‐ATPase on the membrane potential of peritoneal mast cells isolated from male Sprague‐Dawley SPF‐rats. 2 Experiments were performed at 22–26°C in the tight‐seal whole‐cell configuration of the patch‐clamp technique by use of Sylgard‐coated patch pipettes (3–6 MΩ). High‐resolution membrane currents were recorded with an EPC‐9 patch‐clamp amplifier controlled by the ‘E9SCREEN’ software. In addition, a charting programme on another computer synchronously recorded at low resolution (2 Hz) membrane potential and holding current (low‐pass filtered at 500 Hz). 3 Na+/K+‐ATPase activity was measured as the ouabain‐sensitive change in the zero‐current potential. The zero‐current potential in rat peritoneal mast cells measured 2 min after obtaining whole‐cell configuration amounted to 1.7±2.5 mV (n=21). Ouabain (5 mM), a Na+/K+‐ATPase‐inhibitor, had only a very minor effect upon the membrane potential under resting conditions (n=3). 4 When mast cells were superfused with nominal calcium‐free external solution, the cells hyperpolarized (Δ mV: 20.2±3.8 mV (n=5)). In addition, when the mast cells were preincubated in nominal calcium‐free external solution for 12±1.6 min before whole‐cell configuration, the membrane potential amounted to −53.7±9.8 mV (n=8). A subsequent superfusion with ouabain (5 mM) depolarized the membrane potential (ouabain‐sensitive hyperpolarization (Δ mV): 23.0±8.4 mV (n=8)). 5 A high intracellular concentration of Na+ ([Na+]i) (26.6 mM) also resulted in hyperpolarization (Δ mV: 20.2±9.1 mV (n=7)), but only when ATP was present. A subsequent superfusion with ouabain (5 mM) repolarized these cells to −1.2±14 mV (ouabain‐sensitive hyperpolarization (Δ mV): 19.7±7.7 mV (n=7)). 6 The size of the [Na+]i‐dependent hyperpolarization was dose‐dependent. Low [Na+]i (1 mM) had no effect on membrane potential and these cells were unaffected by superfusion with calcium‐free external solution. 7 These data thus directly confirm that the stimulant effect of calcium‐free external solutions on the ouabain‐sensitive changes in the zero‐current potential, and hence the Na+/K+‐ATPase, is mediated through [Na+]i and that the activity of the Na+/K+‐ATPase can have an important influence on the resting membrane potential in rat peritoneal mast cells.

Dependence of histamine release from rat mast cells on adenosine triphosphate

Summaryusing pure populations of rat mast cells, the relation of the ATP content of the cells to histamine release induced by compound 48/80 has been studied. Variable ATP levels in the mast cells have been produced by incubation with appropriate concentrations of oligomycin.The dose-response curves of oligomycin for the inhibition of histamine release and for the reduction in the ATP content of the mast cells are similar. The concentration required for 50% inhibition of histamine release is, however, higher than that for 50% reduction of the ATP level.Comparative study of the reduction of the ATP content and the inhibition of histamine release in samples of the same suspension of mast cells shows a linear relation between 10 to 90% inhibition of histamine release and 40 to 95% inhibition of ATP synthesis.The observations support the hypothesis that ATP is involved in the process of histamine release from mast cells induced by compound 48/80.

Na+‐K + pump activity in rat peritoneal mast cells: inhibition by extracellular calcium

1 Pure populations of rat peritoneal mast cells were used to study cellular potassium uptake. The radioactive potassium analogue, 86rubidium, was used as a tracer for potassium for measurements of the activity of the cellular potassium uptake process. 2 The ouabain‐sensitive and the ouabain‐resistant potassium (86rubidium) uptake of mast cells incubated in the presence of calcium, 1 mmol l−1, were very low, 52 and 147 pmol per 106 cells min−1. 3 Calcium‐deprivation of the cells uncovered a large capacity ouabain‐sensitive potassium (86rubidium) uptake mechanism. The activity of the uptake mechanism was decreased by reintroduction of calcium into the cell suspension, and it was dependent on cellular energy metabolism, temperature and pH. 4 The potassium (86rubidium) uptake of mast cells incubated in a calcium‐free medium occurs through an active and ouabain‐sensitive mechanism that has the nature of an enzyme, and it is mediated by the Na+‐K+ pump located in the plasma membrane. It is demonstrated that the activity of the Na+‐K+ pump mechanism is inhibited by low concentrations of extracellular calcium (0.1–1.2 mmoll−1). The possibility is discussed that calcium‐deprivation may increase the pump activity by increasing the permeability of the plasma membrane for Na+.

IgE‐receptor activated chloride uptake in relation to histamine secretion from rat mast cells

1 Antigen‐stimulated histamine secretion from rat peritoneal mast cells was inhibited when extracellular chloride was replaced by either isethionate or gluconate anions, but the histamine release still remained quite substantial. 2 Rat peritoneal mast cells take up 36Cl and the uptake reaches a steady state after 60 min incubation with the isotope. At steady state, the intracellular chloride level in the cells was calculated to be 29 ± 11.5 mm. 3 The chloride uptake in mast cells was exponential with a rate constant of 0.036 min−1in resting cells. When the cells were stimulated with antigen, and rate constant for chloride uptake increased to 0.90 min−1: an increase of 25 fold. Under identical experimental conditions histamine release increased 3 fold. 4 The rate of chloride uptake in either resting cells or in antigen‐stimulated cells was not changed when the extracellular medium was nominally calcium‐free but histamine release was almost completely inhibited in the absence of extracellular calcium. 5 The putative chloride channel blocker DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid) 0.3 to 30 μm, produced a concentration‐related inhibition of antigen‐stimulated histamine secretion but DIDS (30 μm) did not inhibit the antigen‐stimulated increase of chloride uptake. 6 The cyclic AMP analogue, dibutyryl cyclic AMP (1 mm) produced a delayed increase in chloride uptake in resting mast cells but neither dibutyryl cyclic AMP nor 8‐bromo cyclic AMP per se induced any histamine secretion. 7 Ouabain (1 mm) which inhibits the Na+/K+ATPase in rat peritoneal mast cells, failed to affect the uptake of chloride in resting mast cells. 8 The Na/K/2Cl‐cotransport inhibitor, furosemide (0.7 mm), slowed the unstimulated chloride uptake in resting mast cells and abolished the increased antigen‐induced chloride uptake when added together with antigen. In contrast, spontaneous and antigen‐induced histamine release were unaffected by the presence of furosemide. However, when furosemide was added to the cell suspension 5 min before stimulation, furosemide was without effect on the antigen‐induced chloride uptake. 9 In addition to the chloride uptake mediated by chloride channels which may be related to the mechanism of histamine secretion, crosslinking of the high affinity membrane receptors for IgE is followed by a fast chloride uptake that is likely to occur through a furosemide‐sensitive Na/K/2Cl‐cotransporter.

Adenosine triphosphate levels during anaphylactic histamine release in rat mast cells in vitro. Effects of glycolytic and respiratory inhibitors.

The adenosine triphosphate (ATP) content of rat mast cells was studied during and after anaphylactic histamine release. The almost identical time course of ATP decrease from mast cells treated with either glycolytic or respiratory inhibitors supports the view that the ATP depletion was largely related to the histamine release process and not to an uncoupling of oxidative phosphorylation by an increased concentration of cytosol Ca2+. The ATP content of the cells was not restored within the 2 h of observation. No inhibition of lactate production from mast cells exposed to antigen in the presence of respiratory inhibitors and glucose was observed. Based on the lactate production from mast cells, the turnover time of ATP was calculated to be about 3/4 min.

MECHANISM OF HISTAMINE RELEASE FROM RAT MAST CELLS INDUCED BY THE IONOPHORE A23187: EFFECTS OF CALCIUM AND TEMPERATURE

1 The mechanism of histamine release from a pure population of rat mast cells induced by the lipid soluble antibiotic, A23187, has been studied and compared with data for anaphylactic histamine release reported in the literature. 2 Histamine release induced by A23187 in the presence of calcium 10−3 mol/1 was completed in 10 minutes. By preincubation of the mast cells with A23187 for lOmin in the absence of calcium the histamine release induced by calcium, 10−3 mol/1 or 5 × 10−3 mol/1, was completed in 90s and 45 s, respectively. 3 A23187‐induced histamine release was maximal with calcium 10−3 mol/1 when the cells were incubated at 33 to 39°C for 10 minutes. 4 The cellular mechanism, which was stimulated by A23187 and calcium for the release of histamine, was irreversibly inactivated by incubation at 45°C. 5 An inhibition of energy metabolism was excluded as the cause of the heat inactivation. 6 The dependence of A23187‐induced histamine release on calcium and temperature, the time course of histamine release and the heat inactivation are consistent with the view that the same mechanism is involved in A23187‐induced and anaphylactic histamine release.

The mode of inhibition of the Na+‐K+ pump activity in mast cells by calcium

1 The inhibition by calcium of the Na+‐K+ pump in the plasma membrane of rat peritoneal mast cells was studied in pure populations of the cells by measuring the ouabain‐sensitive uptake of the radioactive potassium analogue, 86rubidium (86Rb+). 2 Exposure of the cells to calcium induced a time‐ and concentration‐dependent decrease in the ouabain‐sensitive K+(86Rb+)‐uptake of the cells without influencing the ouabain‐resistant uptake. The development of the inhibition required the presence of potassium in the medium in the millimolar range (1.5–8.0 mm), and it did not occur at a concentration of potassium (0.24 mm) that is probably rate limiting for the pump activity. In the presence of 1 mM calcium full inhibition developed almost immediately and was not readily reversed. The inhibition was not significantly reduced by 15 min incubation with 1.2 mM EGTA. 3 The inhibitory action of calcium did not develop when the mast cells were incubated in a potassium‐free medium, which is known to block Na+‐K+ pump activity and allow accumulation of sodium inside the cells. Likewise, increasing the sodium permeability of the plasma membrane by monensin abolished the inhibition of the pump activity. In both cases, incubation of the cells with 4.7 mM potassium and tracer amounts of 86Rb+ resulted in a very large uptake of K+(86Rb+) into the cells (up to 2 nmol per 106 cells min−1), indicating a high activity of the Na+‐K+ pump. 4 These observations support the view that long‐term incubation of rat peritoneal mast cells in a calcium‐free medium increases the permeability of the plasma membrane to sodium, and the consequent increase in the intracellular concentration of sodium causes an increase in the activity of the pump. Addition of calcium to the cell suspension decreases the sodium permeability, and hence the pump activity. This hypothesis is supported by the stimulation of pump activity produced by monensin, which is not inhibited by calcium. The enhancement of pump activity after exposure of calcium‐deprived cells to EGTA might be the result of a further increase in the sodium permeability of the plasma membrane.

Calcium requirement in compound 48/80 induced histamine release from rat mast cells in vitro

Abstract The effect of magnesium and EDTA on compound 48 80 -induced histamine release and adenosine triphosphate (ATP) content of mast cells has been studied. Inhibition of histamine release after preincubation of the cells with or without EDTA in the absence of calcium and the reversal by calcium indicate that calcium is required for compound 48 80 -induced histamine release. The presence of magnesium potentiate the inhibition caused by the lack of calcium. The inhibition of histamine release is not related to changes in cellular ATP content. The observations with EDTA suggest that calcium may be provided for the release process from intracellular sources.

Utilization of adenosine triphosphate in rat mast cells during histamine release induced by the ionophore A23187.

1 The rôle of endogenous adenosine triphosphate (ATP) in histamine release from rat mast cells induced by the ionophore A23187 in vitro has been studied. 2 The amount of histamine released by calcium from rat mast cells primed with the ionophore A23187 was dependent on the ATP content of the mast cells. 3 In aerobic experiments a drastic reduction in mast cell ATP content was found during the time when histamine release induced by A23187 takes place. 4 Anaerobic experiments were performed with metabolic inhibitors (antimycin A, oligomycin, and carbonyl cyanide p‐trifluorometroxyphenylhydrazone), which are known to block the energy‐dependent calcium uptake by isolated mitochondria. The mast cell ATP content was reduced during A23187‐induced histamine release under anaerobic conditions in the presence of glucose. This indicates an increased utilization of ATP during the release process. 5 The observations are consistent with the view that energy requiring processes are involved in ionophore‐induced histamine release from rat mast cells although part of the ATP reduction in the aerobic experiments may be due to an uncoupling effect of calcium on the oxidative phosphorylation.